1. Field of the Invention
This invention relates to an improved method for expressing DNA encoding the bone morphogenetic protein-2 family in mammalian cells.
2. Description of Related Art
The disorders associated with bone loss present major public health problems for Western societies. Osteoporosis alone may affect 20 million Americans in the early years of the next century. Hence, there is wide interest in identifying factors or potential therapeutic agents that inhibit bone loss and stimulate the formation of healthy new bone.
Bone is an extremely complex, but highly organized, connective tissue that is continuously remodeled during the life of an adult by cellular events that initially break it down (osteoclastic resorption) and then rebuild it (osteoblastic formation). This remodeling process occurs in discrete packets throughout the skeleton, i.e., in both cortical bone and trabecular bone. It has recently been reported that mouse bone marrow cells can be stimulated to generate osteoclasts in the presence of parathyroid hormone-related protein or vitamin D. See Akatsu et al., Endocrinology, 125: 20-27 (1989); Takahashi et al., Endocrinology, 123: 2600-2602 (1988) and Takahashi et al., Endocrinology, 123: 1504-1510 (1988).
The currently available therapeutic agents known to stimulate bone formation are fluoride, estrogen, metabolites, and vitamin D. Fluoride clearly increases trabecular bone mass, but questions remain about the quality of the new bone formed, the side effects observed in some patients, whether there are beneficial effects on vertebral fracture rates, and whether increased fragility of cortical bone with subsequent propensity to hip fracture follows.
Another approach is using agents that promote resorption (parathyroid hormone) and then interrupt resorption (calcitonin). One proposed, but not validated, such sequential therapeutic regimen is coherence therapy, where bone metabolic units are activated by oral phosphate administration and then resorption is inhibited by either diphosphonates or calcitonin.
Within the past few years several factors that stimulate osteoblasts have been identified in bone, including transforming growth factor-.beta. (TGF-.beta.), fibroblast growth factor, platelet-derived growth factor, insulin-like growth factor-I, and .beta.2 macroglobulin.
Other proteins stored in the bone matrix may also be important for bone formation. When demineralized bone was injected into the muscle or subcutaneous tissue of rats, a cascade of events, including chondrogenesis, ensued. Urist, Science, 150: 893 (1965). Since the 1960s several investigators have attempted to identify and characterize this activity and have provided an assay for purification of such activity. Reddi and Huggins, Proc. Natl. Acad. Sci. USA, 69: 1601-1605 (1972); Sampath and Reddi, Proc. Natl. Acad. Sci. USA. 78: 7599-7603 (1981).
This assay served as the basis for purifying several novel proteins from bone in sufficient quantity and purity to provide amino acid sequence information, including osteogenin, a protein of 22 Kd [Sampath et al., Proc. Natl. Acad. Sci. USA, 84: 7109 (1987); Luyten et al., J. Biol. Chem., 264: 13377-13380 (1989)] and a glycoprotein called osteoinductive factor [Bentz et al., J. Cell. Biol., 107: 162a (1989)]. See also Wang et al., Proc. Natl. Acad. Sci., 85: 9484-9488 (1988). Based on amino acid sequence data, clones encoding several proteins related by sequence similarity to TGF-.beta. were isolated from bovine and human sources. Wozney et al., Science, 242: 1528-1534 (1988); PCT WO 88/00205 published Jan. 14, 1988; U.S. Pat. No. 4,877,864 issued Oct. 31, 1989. These latter proteins included BMP-2A (also known as BMP-2), BMP-2B (also known as BMP-4), and BMP-3. The sequence of tryptic peptides from osteogenin match the sequence reported for BMP-3.
The TGF-.beta. supergene family includes five distinct forms of TGF-.beta. [Sporn and Roberts, in Peptide Growth Factors and Their Receptors, Sporn and Roberts, eds. (Springer-Verlag: Berlin, 1990) pp. 419-472], as well as the differentiation factors vgl [Weeks and Melton, Cell, 51: 861-867 (1987)] and DPP-C polypeptide [Padgett et al., Nature, 325: 81-84 (1987)], the hormones activin and inhibin [Mason et al., Nature, 318: 659-663 (1985); Mason et al., Growth Factors, 1: 77-88 (1987)], the Mullerian-inhibiting substance, MIS [Cate et al., Cell, 45: 685-698 (1986)], the BMPs, and the developmentally regulated protein Vgr-1 [Lyons et al., Proc. Natl. Acad. Sci. USA. 86: 4554-4558 (1989)]. The subset BMP-2A and BMP-2B is approximately 75% homologous in sequence to DPP-C and may represent the mammalian equivalent of that protein.
The proteins of the TGF-.beta. supergene family are disulfide-linked homo- or heterodimers encoded by larger precursor polypeptide chains containing a hydrophobic signal sequence, a long and relatively poorly conserved N-terminal pro region of several hundred amino acids, a cleavage site (usually polybasic), and a shorter and more highly conserved C-terminal region. This C-terminal region corresponds to the processed mature protein and contains approximately 100 amino acids with a characteristic cysteine motif, i.e., the conservation of seven of the nine cysteine residues of TGF-.beta. among all known family members. Although the position of the cleavage site between the mature and pro regions varies among the family members, the C-terminus of all of the proteins is in the identical position, ending in the sequence Cys-X-Cys-X, but differing in every case from the TGF-.beta. consensus C-terminus of Cys-Lys-Cys-Ser. Sporn and Roberts, 1990, supra.
The pro region of TGF-B associates non-covalently with the mature TGF-.beta. dimer [Wakefield et al., J. Biol. Chem., 263: 7646-7654 (1988); Wakefield et al., Growth Factors, 1: 203-218 (1989)], and the pro regions are found to be necessary for proper folding and secretion of the active mature dimers of both TGF-.beta. and activin [Gray and Mason, Science. 247: 1328-1330 (1990)]. The association between the mature and pro regions of TGF-.beta. masks the biological activity of the mature dimer, resulting in formation of an inactive latent form. Latency is not a constant of the TGF-.beta. supergene family, since the presence of the pro region has no effect on activin or inhibin biological activity.
A unifying feature of the biology of the proteins from the TGF-.beta. supergene family is their ability to regulate developmental processes. Regarding bone formation in vivo, of all the proteins in the TGF-.beta. supergene family, the BMPs and TGF-.beta. play the most major role.
Recombinant TGF-.beta.1 has been cloned [Derynck et al., Nature, 316:701-705 (1985)] and expressed in Chinese hamster ovary cells [Gentry et al., Mol. Cell. Biol., 7: 3418-3427 (1987)]. Additionally, recombinant human TGF-.beta.2 [deMartin et al., EMBO J., 6: 3673 (1987)], as well as human and porcine TGF-.beta.3 [Derynck et al., EMBO J., 7: 3737-3743 (1988); ten Dijke et al., Proc. Natl. Acad. Sci. USA, 85: 4715 (1988)], have been cloned. Expression levels of the mature TGF-.beta.1 protein in COS cells are increased by substituting a serine residue for cysteine residues located in the pro region of the TGF-.beta.l precursor. Brunner et al., J. Biol. Chem., 264: 13660-13664 (1989).
BMP-2A and BMP-3 have been recombinantly produced in monkey COS-1 cells and Chinese hamster ovary cells by Wozney et al., supra. However, the level of expression of BMP-2A and -2B cDNA is relatively low when the DNA is not amplified. Higher levels of BMP-2A protein expression in CH cells have been obtained by amplification to a high copy number using methotrexate selection
of dihydrofolate reductase. Wang et al., Proc. Natl. Acad. Sci. USA, 87: 2220-2224 (1990).
Confirmation of the osteogenic activity of BMPs and commercial production thereof depend on the ability to produce useful amounts of active material by recombinant means of expression and development of methods to purify them in an active form. The ability to successfully reconstitute endochondral bone formation remains the standard by which to judge the osteogenic character of candidate factors. The biological activities of BMP-2A, BMP-3, and an unrelated molecule, BMP-1, were originally assessed in an implant model using material expressed in COS cells, resulting in only cartilage formation. Wozney et al., supra. More recently, the partially purified BMP-2A expressed in CHO cells was shown to require a dose of at least 600 ng/implant to induce cartilage and bone formation. Wang et al., 1990, supra. The osteogenic activities of BMP-2B and BMP-3 have not been established.
It is an object of the present invention to provide purified BMP-2B in sufficient quantities to test for its osteogenic activity, and to produce it on a commercial scale.
It is another object to improve the expression levels of BMP-2 DNA in mammalian cells without amplifying the DNA.
It is still another object to achieve higher production of BMP-2 protein than was previously attained at a level of amplification equivalent to that previously employed.
These and other objects will be apparent to those of ordinary skill in the art of molecular biology.